N-Ethylmaleimide-sensitive Fusion Protein Contains High and Low Affinity ATP-binding Sites That Are Functionally Distinct

The ATPase NSF (N-ethylmaleimide-sensitive factor) and its SNAP (soluble N-ethylmaleimide-sensitive factor attachment protein) cofactor constitute the ubiquitous enzymatic machinery responsible for recycling of the SNARE (SNAP receptor) membrane fusion machinery. The enzyme uses the energy of ATP hydrolysis to dissociate the constituents of the SNARE complex, which is formed during the fusion of a transport vesicle with the acceptor membrane. However, it is still unclear how NSF and the SNAP adaptor work together to take the tight SNARE bundle apart. SNAPs have been reported to attach to membranes independently from SNARE complex binding. We have investigated how efficient the disassembly of soluble and membrane-bound substrates are, comparing the two. We found that SNAPs support disassembly of membrane-bound SNARE complexes much more efficiently. Moreover, we identified a putative, conserved membrane attachment site in an extended loop within the N-terminal domain of ␣-SNAP. Mutation of two highly conserved, exposed phenylalanine residues on the extended loop prevent SNAPs from facilitating disassembly of membrane-bound SNARE complexes. This implies that the disassembly machinery is adapted to attack membrane-bound SNARE complexes, probably in their relaxed cis-configuration.All intracellular vesicle transport processes, ranging from secretion in yeast to neurotransmitter release in the brain, depend on the ability of membranes to fuse with each other. Intracellular fusion is mediated by the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) 3 proteins on opposing membranes, which assemble in transconfiguration into four-helix bundle complexes, bringing the membranes into close proximity (for an overview see Refs.

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“…As shown in supplemental Fig. S1, the ATP and NSF requirements were in line with earlier studies (39).…”

Section: Fret and Fluorescence Anisotropy Can Monitor Snare Complex Dsupporting

confidence: 78%

A Conserved Membrane Attachment Site in α-SNAP Facilitates N-Ethylmaleimide-sensitive Factor (NSF)-driven SNARE Complex Disassembly

Winter

Chen

Fasshauer

2009

Journal of Biological Chemistry

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“…This defect in SNAP/ SNARE binding is consistent with an inability of the NSF molecule to attain a binding competent conformation. Such binding incompetence occurs when NSF is in the ADP-bound form (29) and has been seen in mutants that lack or have mutations in the N-domain (27) 2 or that are unable to bind nucleotide in the D1 domain.…”

Section: Depolarization-dependent Phosphorylation Of Nsf Inmentioning

confidence: 99%

“…SNAP-SNARE Complex Binding Assay-The complex formation procedure was modified from our method described previously (29). GSTsyntaxin 1 (cytosolic domain) was incubated with pre-swollen, glutathione-agarose beads (100 g of protein per 100 l of beads) at 4°C in phosphate-buffered saline with 0.01% (v/v) Tween 20, 0.1% (v/v) ␤-mercaptoethanol, and 2 mM EDTA.…”

Section: Methodsmentioning

confidence: 99%

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Phosphorylation of the N-Ethylmaleimide-sensitive Factor Is Associated with Depolarization-dependent Neurotransmitter Release from Synaptosomes

Matveeva¹,

Whiteheart²,

Vanaman³

et al. 2001

Journal of Biological Chemistry

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Critical to SNARE protein function in neurotransmission are the accessory proteins, soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP), and NSF, that play a role in activation of the SNAREs for membrane fusion. In this report, we demonstrate the depolarization-induced, calcium-dependent phosphorylation of NSF in rat synaptosomes. Phosphorylation of NSF is coincident with neurotransmitter release and requires an influx of external calcium. Phosphoamino acid analysis of the radiolabeled NSF indicates a role for a serine/threonine-specific kinase. Synaptosomal phosphorylation of NSF is stimulated by phorbol esters and is inhibited by staurosporine, chelerythrine, bisindolylmaleimide I, calphostin C, and Ro31-8220 but not the calmodulin kinase II inhibitor, Kn-93, suggesting a role for protein kinase C (PKC). Indeed, NSF is phosphorylated by PKC in vitro at Ser-237 of the catalytic D1 domain. Mutation of this residue to glutamic acid or to alanine eliminates in vitro phosphorylation. Molecular modeling studies suggest that Ser-237 is adjacent to an inter-subunit interface at a position where its phosphorylation could affect NSF activity. Consistently, mutation of Ser-237 to Glu, to mimic phosphorylation, results in a hexameric form of NSF that does not bind to SNAP-SNARE complexes, whereas the S237A mutant does form complex. These data suggest a negative regulatory role for PKC phosphorylation of NSF. The molecular mechanisms of neurotransmitter (NT)1 release have been the subject of much attention in recent years resulting in an evolving model known as the SNARE hypothesis (1-3). The SNARE hypothesis holds that membrane proteins in the vesicle (v-SNAREs, e.g. synaptobrevins) bind to a heterodimer in the target membrane (t-SNAREs, heterodimers of syntaxins and SNAP-25-like proteins). v-and t-SNAREs bind to form a 7 S complex (1, 4), composed of a bundle of four parallel, coiled-coil domains (5-8) that, through reconstitution studies, has been demonstrated to be minimally required for bilayer fusion (9). The N-ethylmaleimide-sensitive factor (NSF) and the soluble NSF attachment proteins (SNAPs, not to be confused with SNAP-25) affect the composition and structure of the SNARE complex. SNAPs act as adapters and are required for binding of NSF to the 7 S complex. ATP hydrolysis by NSF causes the resulting 20 S complex to disassemble into monomeric SNAREs (4, 10), a step required for vesicle trafficking (11-13). Detailed kinetic experiments have placed one role of NSF and SNAPs at steps prior to v-/t-SNARE binding (14 -16), suggesting a model in which NSF disassembles cis 7 S complexes that exist in the same bilayer (17-19). The resulting monomeric SNAREs can then form trans 7 S complexes that span the opposing bilayers of the vesicle and target membrane. In this manner, NSF acts as a chaperone to activate or "prime" the SNARE proteins for subsequent trans complex formation and membrane fusion.Studies by Schweizer et al. (20), using NSF-derived peptides microinjected pre-synaptically, showed that ...

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“…The D2 domain is required for hexamerization of NSF (11,(14)(15)(16). The D1 domain contains a high-affinity ATP-binding and hydrolysis site that accounts for the majority of the ATPase activity in NSF (15,17). Recruitment of NSF to SNARE complexes via the SNAP adapters results in the specific stimulation of D1 ATPase activity and disassembly of the SNARE complex (17-20).…”

mentioning

confidence: 99%

SNARE-complex disassembly by NSF follows synaptic-vesicle fusion

Littleton

Barnard

Titus

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

117

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Soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE)-mediated fusion of synaptic vesicles with the presynaptic-plasma membrane is essential for communication between neurons. Disassembly of the SNARE complex requires the ATPase N-ethylmaleimide-sensitive fusion protein (NSF). To determine where in the synaptic-vesicle cycle NSF functions, we have undertaken a genetic analysis of comatose (dNSF-1) in Drosophila. Characterization of 16 comatose mutations demonstrates that NSF mediates disassembly of SNARE complexes after synaptic-vesicle fusion. Hypomorphic mutations in NSF cause temperature-sensitive paralysis, whereas null mutations result in lethality. Geneticinteraction studies with para demonstrate that blocking evoked fusion delays the accumulation of assembled SNARE complexes and behavioral paralysis that normally occurs in comatose mutants, indicating NSF activity is not required in the absence of vesicle fusion. In addition, the entire vesicle pool can be depleted in shibire comatose double mutants, demonstrating that NSF activity is not required for the fusion step itself. Multiple rounds of vesicle fusion in the absence of NSF activity poisons neurotransmission by trapping SNAREs into cis-complexes. These data indicate that NSF normally dissociates and recycles SNARE proteins during the interval between exocytosis and endocytosis. In the absence of NSF activity, there are sufficient fusion-competent SNAREs to exocytose both the readily released and the reserve pool of synaptic vesicles.neurotransmitter release ͉ exocytosis ͉ synapse ͉ Drosophila T he core components of the vesicle-fusion apparatus consist of the plasma-membrane soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein (SNAP) receptors (SNAREs) syntaxin and soluble NSF attachment protein of 25 kDa (SNAP-25) and the vesicle SNARE synaptobrevin͞vesicle-associated membrane protein. Neuronal SNAREs assemble into highly stable SDS-resistant 7S SNARE complexes (1). The ATPase NSF has been implicated in numerous membrane-trafficking steps, including regulated exocytosis (2-8). NSF is recruited to the 7S complex by means of the adapter proteins ␣͞␤-and ␥-SNAP to form a larger 20S complex that is subsequently disassembled by the ATPase activity of NSF (1). Both assembly and disassembly of the SNARE complex are essential for synaptic-vesicle trafficking in vivo (6-10). NSF belongs to the AAA family of ATPases (ATPases with diverse cellular activities) and forms a hollow cylindrical hexamer when viewed with high-resolution electron microscopy (11, 12). The structure of the NSF hexamer depends on the presence or absence of bound ATP or ADP and undergoes substantial conformational changes upon ATP hydrolysis (11). Each monomer consists of three domains (13): an N-terminal domain essential for interaction with the SNAP͞SNARE complex, and two tandem ATPase domains termed D1 and D2. The D2 domain is required for hexamerization of NSF (11,(14)(15)(16). The D1 domain contains a high-affinity ATP-binding and hydrolysis sit...

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N-Ethylmaleimide-sensitive Fusion Protein Contains High and Low Affinity ATP-binding Sites That Are Functionally Distinct

Cited by 72 publications

References 47 publications

A Conserved Membrane Attachment Site in α-SNAP Facilitates N-Ethylmaleimide-sensitive Factor (NSF)-driven SNARE Complex Disassembly

A Conserved Membrane Attachment Site in α-SNAP Facilitates N-Ethylmaleimide-sensitive Factor (NSF)-driven SNARE Complex Disassembly

Phosphorylation of the N-Ethylmaleimide-sensitive Factor Is Associated with Depolarization-dependent Neurotransmitter Release from Synaptosomes

SNARE-complex disassembly by NSF follows synaptic-vesicle fusion

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